Generating immunogenic, nontoxic forms of bacterial toxins (toxoids) is a potentially important application of recombinant DNA technology. The current project has two major goals: (1) to generate an effective recombinant toxoid of Pseudomonas aeruginosa exotoxin A (ETA), and (2) to provide a model system for generating toxoids by mutagenizing active- site residues for cloned toxins. We have used photoaffinity labelling to identify glutamic acid-553 (Glu-553, or E553) as a crucial active- site residue of ETA and employed oligonucleotide-directed mutagenesis to delete this residue or replace it with other amino acids. In continuing these studies we will focus on ETA-E553X, a candidate toxoid formed by specifically deleting Glu-553. We will develop improved methods for producing ETA-E553X and other cloned recombinant antigens in quantity in P. aeruginosa, and perform a more thorough characterization of the biological and biochemical properties of ETA-E553X. This mutant toxin will be subjected to phase 1 safety and immunogenicity trials in humans, and will be used to prepare protein-polysaccharide conjugates with various P. aeruginosa polysaccharide components. We will also generate and characterize other candidate recombinant toxoids of ETA. These studies may have important applications in controlling P. aeruginosa infections; and lessons learned about toxoid construction by mutagenizing active-site residues could be widely applicable.